Aspartase (L-aspartate ammonia-lyase, E.C. 4.3.1.1) catalyzes the reversible deamination of L-aspartic acid to yield fumarate and ammonium ions (or salts). This dearnination reaction is represented by the Formula I: ##STR1##
Many plants and microorganisms produce aspartase to control various biological functions such as allowing microorganisms to grow by using glutamate as the only carbon source. The role of aspartase in this process is to recycle aspartic acid for the regeneration of oxaloacetate as an amino group acceptor.
Aspartase is a tetrameric protein with a subunit molecular weight of about 52,400 Da. Its amino acid sequence of aspartase derived from Escherichia coli (E. coli) has been reported by Takagi et al., Nucleic Acids Res. 13:2063 (1985), to be that described in SEQ ID NO: 1. The similar amino acid sequences of aspartase derived from other organisms have also been reported.
The gene that encodes aspartase, aspA, is a polynucleotide sequence of DNA consisting of approximately 1430 base pairs, including a ribosome binding region and a promoter region. The known sequence of this gene for E. coli is represented in SEQ ID NO: 1.
Aspartase is an allosteric enzyme having tvo structural conformations, a high activity form and a low activity forn. At pH values above neutrality, aspartase must be activated by aspartic acid and divalent metal ions in order to catalyze the addition of an ammonium ion across the double bond of fumarate.
Activation by nonspecific proteolytic cleavage of aspartase is also known. Yumoto et al., Biochim. Biophys. Acta 616:319 (1980) and Yumoto et al., Plysiol. Chem. Phys. 14:391 (1982), for example, have shown that non-specific proteolytic cleavage of peptide bonds near the C-terminal by several different proteases results in a several fold increase of aspartase activity. It was not clear from these studies, however, whether cleavage at a single site or multiple sites was responsible for the enhanced activity and if so, which ones.
It has been observed for the vast majority of enzymes, in contrast to the results observed with aspartase, that the removal of amino acids causes a decrease in the activity of the enzyme. As reported by Sala-Newby et al., Biochim. Biophys. Acta Protein Struct. MoL Enzymol. 1206:155 (1994), for example, the sequential removal of amino acids from the C-terminal of firefly luciferase leads to a stepwise loss of bioluminescent activity without regard to the nature or identity of the amino acid that is removed.
Aspartase is used commercially primarily to produce large quantities of L-aspartic acid. L-aspartic acid is used, for example, to produce polyaspartase for use in detergents and for the enhancement of nutrient absorption in agricultural applications. L-aspartic acid is also used for the manufacture of biodegradable chelating agents and in the food industry as a component of the artificial sweetener aspartame (NutraSweet.RTM.).
Development of a superior enzyme to produce L-aspartic acid faster, cheaper, or both would be a highly desirable improvement over the current art Currently, L-aspartic acid is commercially manufactured using a native or wild-type aspartase enzyme, such as that from E. coli. Heretofore, native aspartase was purified by chromatographic techniques, such as described by Karsten et al., Anal Biochemn. 147:336 (1985). Thus, there is a need to isolate and purify aspartase derivatives with enhanced catalytic activity that can produce as much aspartic acid in a given time with a lower quantity of enzyme.
Only recently has it been discovered that aspartase plays a role in blood clot dissolution. Sjostrom et al., Biochim Biophys. Acta 1324:182 (1997), found that a plasmninogen binding protein, identified as aspartase in this organism, stimulates the activation of plasminogen by tissue plasminogen activator (tPA). tPA is a new drug that has been approved for use in the treatment of heart attacks. This drug accelerates the dissolution of blood clots that lead to heart attacks and strokes. Thus, there is a need to isolate and purify derivatives of aspartase with an enhanced capability to aid in the dissolution of blood clots.